Hypoxia is a central feature of many human diseases, including myocardial infarction, stroke, and cancer. Despite substantial research on cellular responses to hypoxia, our understanding of pathways initiated by low oxygen tension remains, at best, incomplete. A major recent advance in this regard is the discovery of the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF):von Hippel Lindau protein (VHL) pathway. In this pathway, the prolyl hydroxylase PHD (also known as HIF prolyl hydroxylase or EGLN) modifies HIF at two specific prolyl residues, which in turn targets the latter for degradation by the ubiquitin-proteasome pathway in a manner dependent of the VHL E3 ubiquitin ligase complex. The obligatory use of molecular oxygen by PHD in the hydroxylation reaction immediately provides a compelling model by which oxygen tension can be coupled to protein degradation. Thus, under normoxic conditions, HIF is constitutively hydroxylated and degraded. Under hypoxic conditions, HIF is hypohydroxylated, escapes VHL mediated degradation, and then binds to the enhancers of genes involved in adaptation to hypoxia. Indeed, the PHD:HIF:VHL model may even raise the possibility of PHD as a universal oxygen sensor. PHD consist of three isoforms, and current evidence indicates that PHD2 is the key PHD isoform responsible for the maintaining HIF at essentially undetectable levels under normoxia. In the present application, we identify a factor, which we call IOP1, that challenges the notion that the PHD:HIF:VHL pathway is sufficient to account for oxygen sensing. IOP1 binds to PHD2 and is homologous to hydrogenases, iron-containing enzymes with ancestral origins in anaerobic bacteria. Bacterial hydrogenases play central roles in maintaining redox balance under anaerobic conditions. We provide evidence that in mammalian cells, IOP1 can regulate the HIF activity under both normoxic and hypoxic conditions, and that this occurs through the modulation of HIF protein levels. In addition, bacterial iron-containing hydrogenases are well known to be oxygen-labile, raising the intriguing possibility that IOP1 itself may serve as a oxygen sensor. To more fully characterize this novel component of the hypoxia signalling pathway, we will pursue the following specific aims. First, we will determine whether IOP1 regulates the HIF pathway. Second, we will determine the mechanism by which this occurs. Third, we will determine whether IOP1 itself serves as an oxygen sensor. Fourth, we will generate mice with a germline deletion of IOP1 and then characterize both the mice and hypoxic responses in cells derived from these mice. We anticipate that these studies will define a new pathway by which cells respond to hypoxia.